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2021.02.03.429226V1.Full.Pdf bioRxiv preprint doi: https://doi.org/10.1101/2021.02.03.429226; this version posted February 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A modular platform for engineering function of natural and synthetic biomolecular condensates Keren Lasker1,*, Steven Boeynaems2,*, Vinson Lam3, Emma Stainton1, Maarten Jacquemyn4, Dirk Daelemans4, Elizabeth Villa3, Alex S. Holehouse5,6, Aaron D. Gitler2,#, Lucy Shapiro1,# 1Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA 2Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA 3Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093, USA 4KU Leuven Department of Microbiology, Immunology, and Transplantation, Laboratory of Virology and Chemotherapy, Rega Institute, KU Leuven, 3000 Leuven, Belgium 5Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, St. Louis, MO 63110, USA. 6Center for Science and Engineering of Living Systems (CSELS), Washington University in St. Louis, St. Louis, MO 63130, USA *Equal contribution, #emails: [email protected], [email protected] Abstract Phase separation is emerging as a universal principle for how cells use dynamic subcompartmentalization to organize biochemical reactions in time and space1,2. Yet, whether the emergent physical properties of these biomolecular condensates are important for their biological function remains unclear. The intrinsically disordered protein PopZ forms membraneless condensates at the poles of the bacterium Caulobacter crescentus and selectively sequesters kinase-signaling cascades to regulate asymmetric cell division3-5. By dissecting the molecular grammar underlying PopZ phase separation, we find that unlike many eukaryotic examples, where unstructured regions drive condensation6,7, a structured domain of PopZ drives condensation, while conserved repulsive features of the disordered region modulate material properties. By generating rationally designed PopZ mutants, we find that the exact material properties of PopZ condensates directly determine cellular fitness, providing direct evidence for the physiological importance of the emergent properties of biomolecular condensates. Our work codifies a clear set of design principles illuminating how sequence variation in a disordered domain alters the function 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.03.429226; this version posted February 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. of a widely conserved bacterial condensate. We used these insights to repurpose PopZ as a modular platform for generating synthetic condensates of tunable function in human cells. Introduction Biomolecular condensation is a powerful mechanism underlying cellular organization and regulation in cell physiology and disease1,2,8. Many of these condensates are formed via reversible phase separation2,9, which allows for rapid sensing and response to a range of cellular challenges10,11. Biomolecular condensates can adopt a broad spectrum of material properties, from highly dynamic liquids to semi-fluid gels and glasses and solid amyloid aggregates9,12-14. Perturbing protein condensation can alter fitness15-18, and mutations promoting protein aggregation and other pathological phase transitions have been implicated in human disease14,19-23. These observations suggest that the exact material properties of a biomolecular condensate may be important for its function. However, mechanistic links between emergent properties of condensates and cellular/organismal fitness remain largely unexplored. The bacterium Caulobacter crescentus reproduces by asymmetric division24, an event orchestrated by the intrinsically disordered Polar Organizing Protein Z, PopZ3,4. PopZ self-assembles into 200 nm microdomains localized to the cell poles (Fig. 1a) and forms a homogeneous membraneless compartment that excludes large protein complexes, such as ribosomes25,26 (Fig. 1b). In previous work, we found that retention of client proteins in the microdomain is selective for cytosolic proteins that directly or indirectly bind to PopZ, allowing for the spatial regulation of kinase- signaling cascades that drive asymmetric cell division5. PopZ mutants unable to condense into a polar microdomain result in severe cell division defects27. This well-defined and important physiological function of the PopZ microdomain makes it an ideal system to interrogate material property-function relationships in vivo. PopZ phase separates in Caulobacter crescentus and human cells. To probe the dynamic behavior of PopZ, we expressed RFP-tagged PopZ in a strain of Caulobacter bearing the mreBA325P mutation28, which leads to irregular cellular elongation with thin polar regions and wide cell bodies29. While PopZ normally resides at the cell pole, in this background, the microdomain deforms and extends into the cell body before undergoing spontaneous fission, 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.03.429226; this version posted February 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. producing spherical droplets that move throughout the cell (Fig. 1c-d, Supplementary Fig. 1a). The deformation of the microdomain at the thinning cell pole and the minimization of surface tension when unrestrained by the plasma membrane provides in vivo evidence that the PopZ microdomain behaves as a liquid-like condensate. This observation is in line with the partial fluorescence recovery of PopZ upon photobleaching (FRAP), indicating slow internal dynamic rearrangements5 (Fig. 1e). PopZ homologs are restricted to a-proteobacteria, and the sequence composition of the PopZ intrinsically disordered region (IDR) is divergent from the human disordered proteome (Fig. 1f, Supplementary Fig. 1b-e). We reasoned that human cells could serve as an orthogonal system for studying PopZ condensation. When expressed in a human osteosarcoma U2OS cell line, PopZ condensed into micron-sized cytoplasmic condensates (Fig. 1g) that underwent spontaneous fusion events (Fig. 1h) and experienced dynamic internal rearrangements, as assayed by FRAP. Importantly, even though they were expressed in human cells, PopZ condensates retained specificity for their bacterial client proteins, such as ChpT5, and were distinct from human stress granules (Fig. 1i). Thus, PopZ is sufficient for condensation and client recruitment, and human cells serve as an independent platform to study its behavior. PopZ IDR tunes the microdomain viscosity PopZ is composed of three functional regions27,30 (Fig. 2a, Supplementary Fig. 2a): (i) a short N- terminal helical region (H1) used for client binding30,31, (ii) a 78 amino-acid (aa) IDR (IDR-78)31, and (iii) a helical C-terminal region (H2, H3, and H4) which is required for PopZ self- oligomerization27. To define the molecular mechanism driving phase separation of PopZ, we examined the contribution of each of these domains to condensation in human and Caulobacter cells. PopZ mutants missing either the N-terminal region (Δ1-23) or the IDR (Δ24-101) were able to form condensates in both cell types (Fig. 2b). Deletion of the IDR resulted in the formation of irregular gel-like condensates characterized by arrested fusion events in human cells (Fig. 2b) while producing dense microdomains in Caulobacter (Fig. 2b). In contrast, deleting any of the three predicted C-terminal helical regions (Δ102-132, Δ133-156, and Δ157-177) markedly reduced visible PopZ condensates (Fig. 2b). Therefore, the C-terminal helices are required to form condensates, and the IDR appears to play a role in tuning their material properties. 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.03.429226; this version posted February 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Intriguingly, the architecture of the PopZ protein from Caulobacter crescentus is conserved not only within the Caulobacterales order (Fig. 2c), but also across all a-proteobacteria (Supplementary Fig. 2b). Further, despite showing little sequence conservation, the IDR length exhibits a narrow distribution in Caulobacterales with a mean of 93 ± 1 aa (Fig. 2d), while other clades of a-proteobacteria occupy different length distributions (Supplementary Fig. 2c). To characterize the organization and function of the PopZ linker, we performed all-atom simulations. We found the linker adopts an extended conformation, with a radius of gyration (RG) of 34.4 ± 4.8 Å and an apparent scaling exponent (νapp) of 0.7 (Fig. 2e, Supplementary Fig. 3a). Consistent with previous studies32-35, the strong negative charge leads to a self-repulsing polyelectrolyte, driving expansion beyond the denatured limit and to a tight coupling between the linker length and its global dimensions (Fig. 2h). These results suggest that IDR length is constrained across species. To test the effect of altering IDR length on its condensation, we generated PopZ mutants with a truncated or expanded IDR; namely, IDR-40, corresponding to half the wildtype IDR length and an IDR-156, corresponding to
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